Herpes simplex virus (HSV) is a pathogen affecting about 80-90% of the general population. The prevalence of aggressive intraoral and extraoral recurrent herpetic infections is increasing and is exacerbated by increasing numbers of immunocompromised patients. This rising prevalence is due, in part, to the development of drug-resistant HSV which poses an obvious threat clinically. This clinical problem is a recently recognized phenomena and is a subject of concern. The rate at which drug-resistant mutants are generated is largely determined by the fidelity of viral DNA replication. HSV DNA polymerase (Pol) is the central replicative protein and is pivotal in determining replication fidelity. Thus, study of the HSV Pol and its related functions on the fidelity of DNA replication will aid in understanding the mechanisms of the development of drug-resistant mutants. In addition, HSV DNA replication provides an excellent and convenient model for the study of the eukaryotic DNA replication. Therefore, this study will also provide information on the fidelity of eukaryotic DNA replication detailed at the sequence level. The first aim is to study the contribution of various catalytically important regions of HSV Pol on the fidelity of DNA replication. In vivo assay by using various HSV pol mutants and the newly developed mutagenesis shuttle plasmid, the relative contribution of various Pol catalytic regions in determining the fidelity of DNA replication will be assessed. This assay allows the analysis of the precise nature of mutations at the sequence level. The second aim is to study the contribution of the 3'-5' exonucleolytic activity intrinsic to HSV-1 Pol on the fidelity of DNA replication. Assays will be performed in vitro to examine the exonuclease activity, the proofreading and the related fidelity, using purified wild type and mutated Pols containing mutations within various functional regions, especially the exonucleolytic region. The third aim is to examine the contribution of a Pol accessory protein, UL42, on the fidelity of DNA replication. UL42 has been shown to enhance the processivity of Pol and is a potential antiviral target. Both in vivo and in vitro assays, using various Pol and UL42 mutants which affect their activities, will be assessed. These studies will provide a better understanding of the structure- function relationship of HSV Pol and the mechanisms by which drug- resistant HSV mutants arise, and may provide useful information for future strategy in the development of the novel antiviral therapy.